A comparison of 64Cu-labeled bi-terminally PEGylated A20FMDV2 peptides targeting integrin ανβ6

Expression of epithelial-specific integrin ανβ6 is up-regulated in various aggressive cancers and serves as a prognostic marker. Integrin-targeted PET imaging probes have been successfully developed and tested in the clinic. Radiotracers based on the peptide A20FMDV2 derived from foot-and-mouth disease virus represent specific and selective PET ligands for imaging ανβ6-positive cancers. The present study aims to describe the radiolabeling, in vitro and in vivo evaluation of a bi-terminally PEGylated A20FMDV2 conjugated with DOTA or PCTA for 64Cu radiolabeling. Stability studies showed radiolabeled complexes remained stable up to 24 h in PBS and human serum. In vitro cell assays in CaSki cervical cancer cells and BxPC-3 pancreatic cancer cells confirmed that the peptides displayed high affinity for αvβ6 with Kd values of ~50 nM. Biodistribution studies revealed that [64Cu] Cu-PCTA-(PEG28)2-A20FMDV2 exhibited higher tumor uptake (1.63 ± 0.53 %ID/g in CaSki and 3.86 ± 0.58 %ID/g in BxPC-3 at 1 h) when compared to [64Cu]Cu-DOTA-(PEG28)2-A20FMDV2 (0.95 ± 0.29 %ID/g in CaSki and 2.12 ± 0.83 %ID/g in BxPC-3 at 1 h) . However, higher tumor uptake was accompanied by increased radioactive uptake in normal organs. Therefore, both peptides are appropriate for imaging ανβ6-positive lesions although further optimization is needed to improve tumor-to-normal-tissue ratios.


INTRODUCTION
Integrins are a class of receptors that play essential roles in mediating cell adhesion, making transmembrane connections to the cytoskeleton, and modulating many intracellular signaling pathways [1,2]. Because of their diverse functions, integrins have been studied extensively for decades, leading to the design and development of integrin antagonists in the treatment of multiple types of cancers. In recent years, α ν β 6 has received much attention where its overexpression has been discovered to promote malignant behavior and tumor progression, resulting in poor prognosis and a striking reduction in survival rates for cancer patients [3][4][5][6][7]. α ν β 6 has many regulatory functions in oncogenesis and its interaction with fibronectin and/or activation of TGFβ1 are known to lead to invasion and cancer migration [8,9]. Increased expression levels of α ν β 6 have been shown in various cancers including pancreatic, cervical, non-small cell lung cancer and oral squamous cell carcinoma [10][11][12][13].
This study focuses on the 20-amino-acid, A20FMDV2, derived from the foot-and-mouth disease virus, which was used as a first-generation radiotracer for targeting α ν β 6 in vivo [14]. A20FMDV2 was initially labeled with 18 F and showed specific binding to α ν β 6 both in in vitro cell assays and in vivo tumor-bearing models [18][19][20]. Though the initial lead compound A20FMDV2 exhibited good affinity towards integrin α ν β 6 , in vivo studies in tumor mouse models showed rapid excretion, metabolic breakdown, and unexpected high and persistent levels of radioactivity in nontarget organs. To improve the pharmacokinetics of peptides, modifications such as cysteine amino acid substitutions, cyclization, PEGylation and incorporation of non-proteinogenic amino acid substitutes have been introduced [21][22][23]. Bi-terminal PEGylation of the peptide resulted in favorable in vitro and in vivo behavior concerning α ν β 6 cell binding affinity and tumor uptake in mouse models [23]. This led to the first-in-human microdose study to assess the safety and pharmacokinetics of [ 18 F]FBA-(PEG28) 2 -A20FMDV2 which confirmed the favorable performance of biterminally PEGylated peptide for identification of small lesions in primary sites as well as common sites of metastatic diseases [24].

Macrocyclic chelator integrin α ν β 6 targetting peptides
Bi-terminally PEGylated A20FMDV2 were custom synthesized and conjugated with DOTA and PCTA ( Figure 1) and found to be >93 % pure by HPLC and confirmed by mass spectrometry.

Radiochemistry
The radiolabeling results of the peptides with 64 Cu is shown in Table 1. Radiolabeled products were obtained in high radiochemical purity (>95%) as determined by radio-HPLC and used without further purification. The molar activity of both radiolabeled peptides was 18 Figure 1). Similarly, radio-TLCs demonstrated more than 95% radiochemical purity (Supplementary Figure 2).

Serum stability
In vitro stability studies of all radiotracers were assessed by incubating in PBS 1X and human serum. There was less than 4% loss of 64 Cu in PBS or human serum observed at 24 h (Supplementary Figure 3).

Cell binding and internalization
Both radiolabeled peptides showed binding to α ν β 6expressing cell lines that were significantly inhibited by blocking with A20FMDV2, demonstrating α ν β 6 -specific binding ( Figure 2

Radioligand saturation binding
Receptor-binding affinity of the radiolabeled peptides to α ν β 6 was evaluated on CaSki and BxPC-3 cell lines using a saturation binding assay at 4°C. Binding affinities were investigated by determining the equilibrium dissociation constant (K d ) and the maximum binding capacity (B max ) of radiolabeled conjugates to integrin α ν β 6 -positive cells. Representative saturation binding curves in CaSki and BxPC-3 cell lines are shown in Figure 3.

DISCUSSION
Integrin α ν β 6 , expressed exclusively in epithelial cells, lends itself to be an excellent molecular target for imaging and therapy as it is not expressed in normal adult epithelia but is expressed under special wound healing conditions and in cancer [3][4][5][6][7]. In fact, overexpression of integrin α ν β 6 has been observed in various forms of cancer including those of pancreatic, cervical, non-small cell lung cancer and oral squamous cell carcinoma [10][11][12][13]. Previous research has shown the correlation between α ν β 6 expression with more aggressive disease, reduced survival rates and increased chance of metastasis. Therefore, the design and development of α ν β 6 -targeting peptides that can serve as both imaging and therapeutic agents would be valuable. In this regard, the 20-mer peptide, A20FMDV2, has been extensively studied due to its specific binding toward integrin α ν β 6 [14]. The lead peptide was optimized and radiolabeled with 18 F for imaging with a bi-terminal PEG construct used for first-in-human studies [24]. The purpose of this study was to evaluate A20FMDV2 for radiolabeling with copper for potential imaging or   therapy. Previous studies investigated 64 Cu labeling, only with mono-PEGylation, which is not optimal [25,26]. Therefore, this study employed the bi-terminally PEGylated peptide conjugated to DOTA or PCTA for radiolabeling with 64 Cu. The two peptides are compared in vitro and in vivo.
All radiotracers were radiolabeled in good radiochemical purity (>95 %) at a molar activity of 18.  [25,26]. This indicates that the introduction of another PEG28 does not affect the complexation of 64 Cu with DOTA. Labeling of the PCTA peptide at 45°C resulted in 72% labeling; therefore we increased the temperature to 80°C for 15 mins to achieve >95% labeling. It is not clear why higher temperatures are needed as other studies have showed efficient 64 Cu-labeling of PCTA conjugates at room temperature [27,28], while some have used temperatures >60°C [29,30]. While the in vitro serum stability cannot predict in vivo degradation from the liver and other organs, it can be used as a first screen and showed that both peptides were stable enough to warrant further in vivo investigation.
Similar to Hu et al., more than 75% of bound radioactivity of the two chelates were internalized by 1 h in both CaSki and BxPC-3 cells [25]. While the total amount of bound radioactivity at 1 h was less than 60% observed by Hu et al. for the [ 64 Cu]Cu-DOTA labeled A20FMDV2 peptide, this could be due to the use of different cell lines or the additional PEG28 in our studies  [25]. The saturation binding showed good affinity of 30-60 nM for both peptides when evaluated in CaSki or BxPC-3 cells. This is generally lower than the reported 1.73 ± 0.46 nM for [ 111 In]In-DTPA-A20FMDV2, which can be due to the introduction of PEGylation onto the original peptide [31]. The B max values for BxPC-3 were twice as higher for CaSki cells, which is supported by the higher tumor uptake of radiotracers in BxPC-3 compared to CaSki tumor models.
Overall, uptake in normal organs of [ 64 Cu]Cu-PCTA-(PEG28) 2 -A20FMDV2 was generally higher than that of [ 64 Cu]Cu-DOTA-(PEG28) 2 -A20FMDV2 at the earlier time points (1 and 4 h), especially in the kidney. The increased kidney uptake of [ 64 Cu]Cu-PCTA-(PEG28) 2 -A20FMDV2 may be due to its +2 charge compared to the +1 charge of [ 64 Cu]Cu-DOTA-(PEG28) 2 -A20FMDV2. Cationic portions of peptides may form electrostatic interactions with negative surface charge of proximal tubular cells of kidney, resulting in the trapping of the radiotracers and their metabolites in the tubular cells [32,33]. Higher uptake in other tissues for [ 64 Cu]Cu-PCTA-(PEG28) 2 -A20FMDV2 and in particular the liver may be due to the higher lipophilicity of PCTA compared to DOTA. In addition, there may be expression of α ν β 6 in normal organs such as the lung, kidney, muscle, and heart due to inhibition of radiolabeled peptide uptake by an excess of unlabeled peptide (Supplementary Tables 3  and 4). Previous pre-clinical studies have not investigated expression in normal organs, however, clinical evaluation of similar peptides has shown low uptake in the brain, bone, liver, and lung indicating the potential value of imaging metastases in these sites.
In Thus, both peptides have similar tumor to normal tissue ratios and would be equally good for imaging and therapy, which contradicts our hypothesis that [ 64 Cu]Cu-PCTA-(PEG28) 2 -A20FMDV2 would be superior. Future work will focus on modifications of the peptides to improve affinity and tumor to normal tissue ratios by possibly cyclizing the peptide and substituting more hydrophilic amino acids in positions that do not adversely affect binding.

General information
All solvents and reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) or Fisher Scientific (Pittsburgh, PA, USA) and used as received unless stated otherwise. All solutions and buffers were prepared using HPLC-grade water. Peptides were custom synthesized and characterized by AnaSpec (Fremont, CA, USA). Stock solutions of the peptides (1 nmol/µl) were prepared in HPLC-grade water and stored at −20°C before use. Non-PEGylated A20FMDV2 peptide served as blocking agent. Radio-TLCs employed Whatman 60 Å silica gel thinlayer chromatography (TLC) plates and were analyzed using a Bioscan 200 imaging scanner (Bioscan, Inc., Washington, DC, USA). Reversed-phase high-pressure liquid chromatography (HPLC) was used to evaluate the radiolabeling efficiency. HPLC utilized a two-solvent system: water (0.05% trifluoroacetic acid (TFA)) and acetonitrile (0.05% TFA). The system was equipped with UV absorbance detectors (UV, 220 and 280 nm), a NaI radiotracer detector and a photodiode array detector. HPLC analysis of peptides used Kinetex (Phenomenex) C-18 column (5 μm, 4.6 × 150 mm I.D.).

Radiochemical synthesis of [ 64 Cu]Cu-DOTA-(PEG28) 2 -A20FMDV2 and [ 64 Cu]Cu-PCTA-(PEG28) 2 -A20FMDV2
64 Cu was produced from 64 Ni(p,n) 64 Cu nuclear reaction on enriched 64 Ni on a TR-19 biomedical cyclotron (Advanced Cyclotron Systems, Inc. -Canada) at Mallinckrodt Institute of Radiology, Washington University School of Medicine, and purified with an automated system using standard procedures [34,35]. The resulting activity was diluted in 0.1M HCl at a specific activity ranging from 300 to 2000 mCi/μg. An aliquot of 2 µl DOTA-(PEG28) 2 -A20FMDV2 peptide (2 nmol) was diluted to 100 µl with 0.1M NH 4 OAc (pH 5.5). A stock solution of 64 Cu in 0.1M HCl was diluted ten-fold with 0.1M NH 4 OAc (pH 5.5) for radiolabeling and 1mCi of 64 Cu was added to the reaction mixture. The reaction mixture was incubated at 45°C for 1 h. The product was evaluated for radiochemical purity by radio-HPLC and TLC with a mobile phase of 50 mM DTPA. The radiolabeled complex remained at the origin in the TLC system, while the free 64 Cu moved with solvent front.

Serum stability
100 μl of human serum albumin (HSA 1g/ml) or 1X Phosphate Buffered Saline (PBS) was prepared in Eppendorf tubes, followed by the addition of 100 μl of www.oncotarget.com 64 Cu-radiolabeled peptides. The resulting reaction mixtures were incubated at 37°C with moderate agitation. At time points of 1, 4 and 24 h, aliquots (0.5 µl) were withdrawn from the sample and spotted on TLC plates. Radio-TLCs with a mobile phase of 50 mM diethylenetriamine pentaacetate (DTPA) was performed to evaluate the fraction of intact radiotracer at specific time points.

Flow cytometry
Wash buffer of PBS 1X, 0.1% BSA, and 0.1% sodium azide was prepared. Confluent cells were harvested and re-suspended in wash buffer. Aliquots of 3 × 10 5 cells in 96-well-plate were incubated with 100 µl primary Ab 10D5 (α ν β 6 positive) (10 µg/mL in FACS buffer) at room temperature for 1 h, 500 rpm. After washing the cells three times with wash buffer, cells were incubated in 100 µl of Alexa Fluor 488 goat-anti-mouse (10 µg/mL diluted to 1:50 in FACS buffer) for 30 mins at room temperature. Cells were then rinsed and re-suspended in a final volume of 200 µl wash buffer. In control samples, the primary Ab 10D5 antibody was replaced by IgG2a Isotype Control (α ν β 6 negative). Flow cytometric analysis was performed using a FACScan flow cytometer and data presented as mean fluorescence units (MFUs). . Assay tubes were pretreated with 5% bovine serum albumin (5% wt/v in PBS) to block non-specific binding. Reaction mixtures were incubated at various time points (15 mins, 30 mins, 1 h, 2 h, 4 h and 6 h) with moderate shaking to prevent cell settling. Cells were centrifuged and supernatant was aspirated from cell pellets. The pellets underwent additional washes with 0.5 mL ice-cold PBS (3 times) and the fraction of bound radioactivity was measured with a gamma counter. To determine the fraction of internalized radiotracers, cells were treated with 2 × 0.5 mL of 20 mM sodium acetate (pH 4.0) for 5 mins to remove surfacebound radioactivity. Following centrifugation, cells were washed with 0.5 mL ice-cold PBS and counted for activity. To determine the specific binding towards α ν β 6 , non-PEGylated peptide (20 μg) was added 10 mins before the addition of radiotracers as a blocking agent. The experiments were performed in triplicate.

Radioligand saturation binding
A cell suspension of 1.0 × 10 8 , either CaSki or BxPC-3 cells in 5 mL PBS was prepared. The non-PEGylated peptide was prepared to 10 µg/100 µl in PBS 1X as a blocking agent. Assay tubes were pretreated with 5% bovine serum albumin (5% wt/v in PBS) to block non-specific binding. A solution of 100 µL cells (2 × 10 6 cells) and 100 µl of blocking or PBS were added to each assay tube, followed by a series of 64 Cu-radiolabeled peptide concentrations that ranged between 5 nM and 1000 nM. Tubes were slightly agitated and placed on ice for 1 h. After incubation, tubes were centrifuged for 3 mins, and the supernatant was removed. Cell pellets were washed with ice-cold PBS three times and measured for activity on a gamma counter to determine the amount of surface-bound radioactivity. The experiments were done in triplicate in the presence and absence of a 1000-fold excess blocking agent. Saturation binding curves were generated with x-axis as the molarity of radiolabeled peptides vs. y-axis as specific binding in fmol. Curves were fitted with GraphPad Prism 9 to find the equilibrium constant (K d ) and the maximum binding capacity (B max ).

Biodistribution studies
Animals were supplied by Charles River Laboratories (Wilmington, MA, USA), and were handled in compliance with the Guidelines for Care and Use of Research Animals established by the Division of Comparative Medicine and the Animal Studies Committee of Washington University School of Medicine under protocol #20-0214. Female athymic nude mice were inoculated on the right flank with 10 × 10 6 CaSki or 5 × 10 6 BxPC-3 cells. Tumors were allowed to grow until they approached approximately 100 mm 3 in volume. Radiolabeled peptides were diluted in saline to a dose of 0.37 MBq (10 µCi) per 100 µl. Animals were injected intravenously with 0.37 MBq (10 µCi) of [ 64 Cu]Cu-DOTA-(PEG28) 2 -A20FMDV2 or [ 64 Cu]Cu-PCTA-(PEG28) 2 -A20FMDV2 peptides and sacrificed by cervical dislocation at 1, 4, and 24 h. For blocking experiments, non-PEGylated peptide (100 µg in 100 µL saline) was injected ten minutes before radiotracer. Blood, lung, liver, spleen, kidney, muscle, heart, bone, and tumor were harvested. The amount of radioactivity in each organ was determined by gamma counting, and the percent injected dose per gram of tissue (% ID/g) was calculated.

PET/CT imaging
BxPC-3 cells (5 × 10 6 ) were inoculated in the right shoulder of female athymic nude mice 3-4 weeks before PET imaging sessions. All the experiments involving animals followed the Guidelines for Care and Use of Research Animals established by the Division of Comparative Medicine and the Animal Studies Committee of Washington University School of Medicine under protocol #20-0214. On the day of the experiment, mice were injected intravenously with 3.7 MBq (100 µCi) of [ 64 Cu]Cu-DOTA-(PEG28) 2 -A20FMDV2 or [ 64 Cu] Cu-PCTA-(PEG28) 2 -A20FMDV2 peptides. Mice were imaged with CT followed by static PET scans at 1, 4, and 24 h after tracer administration on an Inveon small animal PET/CT scanner (Siemens Medical Solutions, Malvern, PA, USA). Static images were collected for 20 min and reconstructed with the maximum a posteriori (MAP) reconstruction algorithm using the Inveon Reseach Workstation image display software (Siemens). Regions of interest (ROI) were selected based on co-registered anatomical CT images, and the associated radioactivity was measured using the Inveon software. The maximum standard uptake value (SUV) was calculated as the highest regional radioactivity concentration (nCi/cc) × animal weight (g)/decay-corrected amount of injected dose (nCi).

Statistical analysis
Quantitative data were processed by Prism 9 (GraphPad Software, La Jolla, CA, USA) and expressed as Mean ± SD. Statistical analysis was performed using oneway analysis of variance and Student's t-test. Differences at the 95% confidence level (p < 0.05) were considered statistically significant.